The long-term goal of this project is to understand the chemical basis of transcriptional activation from class II promoters in eukaryotic cells. This phenomenon is at the heart of many genetic programs including embryonic development. Some oncogene products are now known to be oncogenic versions of transcriptional control factors; therefore, an appreciation of transcriptional activation mechanisms is crucial to understanding certain cancers. In this proposal, we focus on structurally characterizing several transcriptional activation domains of the acidic variety. These regions of transcriptional activation proteins contact the basal transcriptional machinery either directly or through co-activators to stimulate gene expression. Polypeptide fragments corresponding to the activation domains of the herpes simplex virus VP16 transsactivator, the yeast GCN4 protein and the yeast GALA protein, as well as interesting mutants, will be expressed and purified. Circular dichroism and two-dimensional NMR spectroscopy will be used to characterize the solution structure of these polypeptides. The target fragments (save one) range in size from 19 to 78 amino acids, well within the range of modem NMR methodology. Since the activation domains chosen for analysis vary widely in their in vivo potency, these studies will not only address the question of whether a conserved structure exists for acidic activation domains, but will also allow a correlation between structure and function. Protein affinity chromatography will be used to address the question of what part of the transcriptional machinery is contacted by the GAL4 activation domains. Variations of this experiment using cloned GAL80 protein, a negative regulatory factor that binds to one of the activation domains of GAL4, will be employed to address the question of how activation domains are silenced when appropriate. In summary, these experiments will provide direct answers to key questions regarding the structure and biochemical properties of acidic transcriptional activation domains.